While non-reactivability of cholinesterase from their phosphyl conjugates (aging) is attributed to an unimolecular process involving loss of alkyl group from the phosphyl moiety, no conclusive evidence is available that this is the only reaction path and involvement of other post-inhibitory processes cannot be ruled out, To address this issue, molecular masses of the bacterially expressed recombinant human acetylcholinesterase and of its conjugates with a homologous series of alkyl methyl-phosphonofluoridates, were measured by electrospray-ionization mass spectrometry (ESI-MS). The measured mass of the free enzyme was 64700 De (calculated 64 695 Da) and those of the methylphosphono-HuAChE adducts, bearing isopropyl, isobutyl, 1,2-dimethylpropyl and 1,2,2-trimethylpropyl substituents, were 64 820, 64 840, 64 852 and 64 860 Da, respectively. These values reflect both the addition of the phosphonyl moiety and the gradual mass increase due to branching of the alkoxy substituent, The composition of these adducts change with time to yield a common product with molecular mass of 64780 Da which is consistent with dealkylation of the phosphonyl moieties, Furthermore, in the case of 1,2,2-dimethylpropyl methylphosphono-HuAChE, the change in the molecular mass and the kinetics of non-reactivability appear to occur in parallel indicating that dealkylation is indeed the predominant molecular transformation leading to 'aging' of phosphonyl-AChE adducts.